DESCRIPTION: The revised proposal continues to be based on the hypothesis that Ca2+ and Pi released locally during bone remodeling induce apoptosis of proximal osteoblasts. There are two specific aims proposed: 1) to measure the effect of Ca2+ and Pi on apoptosis of osteoblast-like cells in culture, including studies to confirm the mechanism of cell death and examine the ability of ion uptake inhibitors to promote survival; and 2) to determine if Ca2+ and Pi initiate apoptosis in osteoblasts by inducing a mitochondrial membrane permeability transition (MMPT). The latter will be accomplished using fluorescent probes that separately measure changes in membrane voltage, intracellular Ca+2, and caspase-3 activation. While pursuing these studies, the candidate will participate in weekly laboratory meetings, attend departmental and university seminars, present in journal club, and attend selected course lectures on areas related to his training at the University of Pennsylvania School of Dental Medicine. The revised application includes new approaches to improve quantitation of experimental findings, a criticism relayed to the applicant in the previous critique.